Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes

Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,     

Temperature dependence of endocytosis mediated by the asialoglycoprotein receptor in isolated rat hepatocytes. Evidence for two potentially rate-limiting steps

The rate of endocytosis of cell surface-bound [3H]-asialo-orosomucoid was determined as a function of temperature. Freshly isolated rat hepatocytes were allowed to bind [3H]asialo-orosomucoid at 4 degrees C, washed to remove nonbound ligand, and internalization was then assessed by the resistance of cell-associated radioactivity to release by the Ca2+ chelator EDTA. At 10 degrees C or below, endocytosis is negligible. Above 10 degrees C, the rate of endocytosis is proportional to temperature but the increase of the rate of endocytosis with increasing temperature changes sharply at about 20 degrees C. From 10-20 degrees C, the apparent activation energy for endocytosis, calculated from an Arrhenius plot, is 45.9 kcal/mol and the temperature coefficient, Q10, is 15.6. However, between 20 and 41 degrees C, the calculated activation energy is 17.0 kcal/mol and the Q10 is 2.6. Although the rate of endocytosis of previously bound [3H]asialo-orosomucoid is very dependent on the temperature, the final extent of endocytosis is essentially temperature-independent between 14 and 37 degrees C. The results suggest that there are at least two steps in the overall process of endocytosis mediated by the asialoglycoprotein receptor on isolated hepatocytes which can be potentially rate-limiting, one at 10 degrees C and another at approximately 20 degrees C.     

The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes

The function of intracellular asialoglycoprotein receptors during the endocytosis of asialo-orosomucoid in isolated hepatocytes was assessed by following changes in the occupancy of intracellular receptors. Unoccupied total cellular (inside and surface) or surface receptors were quantified at 0 degrees C by the binding of 125I-asialo-orosomucoid in the presence or absence, respectively, of digitonin. Freshly isolated cells had about 17% of their total receptors on the surface. After incubation at 37 degrees C, the receptor distribution changed to 25 to 50% on the cell surface and 50 to 75% inside the cell. At 37 degrees C, the average total number of receptors/cell was 4.5 x 10(5). Dissociation constants, determined from equilibrium binding studies in the presence or absence of digitonin to assess total or surface receptors, were identical (5.4 +/- 1.4 and 5.6 +/- 1.1 x 10(-9) M, respectively). In the presence of asialo-orosomucoid at 37 degrees C, there was both a time- and a concentration-dependent decrease in surface and intracellular receptor activity. This receptor activity decrease was reversed by removing asialo-orosomucoid from the medium or by washing the digitonin-permeabilized cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prior to quantification of receptor activity. Within 1 to 2 h in the presence of excess asialo-orosomucoid, a steady state was attained in which approximately 70% of the intracellular receptors were occupied. The kinetics of receptor activity recovery on the cell surface after internalization of a pulse of ligand is different than the rate of recovery of internal receptor activity. The results suggest that all of the internal asialoglycoprotein receptors are functional and participate during endocytosis. Internal receptors may be functionally equivalent to those on the surface or they may serve a reservoir or routing function for internalized ligand.     

The effect of staurosporine, a protein kinase inhibitor, on asialoglycoprotein receptor endocytosis

Receptor-mediated endocytosis via coated pits is modulated by the activity of protein kinases and protein phosphorylation. We examined the effects of the potent protein kinase inhibitor staurosporine (SSP) on endocytosis of the asialoglycoprotein (ASGP) receptor in HepG2 cells. Staurosporine caused a rapid (<2 min) inhibition of ligand internalization from the cell surface. In contrast the rate of receptor exocytosis from intracellular compartments to the cell surface was not altered (t_1/2 = 8 min). This resulted in increased ASGP receptors at the plasma membrane (140% of control) while the total number of receptors per cell was unchanged. Receptor up-regulation was half-maximal at 30 nM SSP. At this concentration staurosporine also inhibited the internalization of iodinated transferrin by HepG2 cells and SK Hep-1 cells, another human hepatoma-derived cell line. Staurosporine was without effect on the non-receptor-mediated uptake of Lucifer yellow by pinocytosis. We investigated the possible involvement of protein kinase C in the inhibitory effects of staurosporine on receptor endocytosis. The active protein kinase C inhibitor H7 did not inhibit ASGP receptor internalization. Furthermore depletion of cellular protein kinase C by overnight incubation with 1 μM phorbol myristate acetate did not abrogate the SSP effect. Together these data suggest that the mechanism of SSP action is independent of the inhibition of protein kinase C. In conclusion staurosporine is a potent and rapid inhibitor of receptor trafficking which is specific for receptor internalization from the plasma membrane.     

Microtubule-depolymerizing agents inhibit asialo-orosomucoid delivery to lysosomes but not its endocytosis or degradation in isolated rat hepatocytes

Microtubule-depolymerizing drugs, such as colchicine, vinblastine sulfate, colcemide and podophyllotoxin, cause an apparent inhibition of the ability of rat hepatocytes to degrade asialo-orosomucoid. However, the binding of asialo-orosomucoid to the cell surface at 0 degrees C, the endocytosis of pre-bound glycoprotein at 37 degrees C, and the dissociation of internal receptor-glycoprotein complexes are unaffected by these microtubule drugs. Receptor recycling is slowed but still occurs, although degradation is blocked. The rate of degradation is decreased by low concentrations of drugs. (For example, 0.25 microM vinblastine sulfate, colchicine and colcemide inhibited 93%, 79% and 26%, respectively.) Neither beta- nor gamma-lumicolchicine affected any of the processes examined. The degree of inhibition with colchicine could be enhanced by a brief treatment of the cells at low temperature to depolymerize microtubules. However, if cells were allowed to endocytose asialo-orosomucoid at 37 degrees C prior to addition of the microtubule drug, then the inhibition of protein degradation was greatly reduced. The decrease in the inhibition of degradation was proportional to the amount of time that cells were exposed to asialoglycoprotein before addition of the drug. The results indicate that the segregation of protein from receptor after they dissociate and/or the subsequent translocation of internalized asialoglycoprotein from the cell perimeter to the lysosomal region requires intact microtubules.     

Microtubule-depolymerizing agents inhibit asialo-orosomucoid delivery to lysosomes but not its endocytosis or degradation in isolated rat hepatocytes

Microtubule-depolymerizing drugs, such as colchicine, vinblastine sulfate, colcemide and podophyllotoxin, cause an apparent inhibition of the ability of rat hepatocytes to degrade asialo-orosomucoid. However, the binding of asialo-orosomucoid to the cell surface at 0°C, the endocytosis of pre-bound glycoprotein at 37°C, and the dissociation of internal receptor-glycoprotein complexes are unaffected by these microtubule drugs. Receptor recycling is slowed but still occurs, although degradation is blocked. The rate of degradation is decreased by low concentrations of drugs. (For example, 0.25 μM vinblastine sulfate, colchicine and colcemide inhibited 93%, 79% and 26%, respectively.) Neither β- nor γ-lumicolchicine affected any of the processes examined. The degree of inhibition with colchicine could be enhanced by a brief treatment of the cells at low temperature to depolymerize microtubules. However, if cells were allowed to endocytose asialo-orosomucoid at 37°C prior to addition of the microtubule drug, then the inhibition of protein degradation was greatly reduced. The decrease in the inhibition of degradation was proportional to the amount of time that cells were exposed to asialoglycoprotein before addition of the drug. The results indicate that the segregation of protein from receptor after they dissociate and/or the subsequent translocation of internalized asialoglycoprotein from the cell perimeter to the lysosomal region requires intact microtubules.     

Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines

We investigated whether the folate receptor α-isoform (FRα), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form of folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FRα (COR ≫ OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0° and 37°C. The amount of 5-methyl[³H]THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FRα expression, e.g., OVCAR4, showed efficient internalization. Time-course studies indicated that, whereas no uptake was detected at 0°C, at 37°C the internalized fraction showed a slow and constant increase, until 4 h. At this time, the internalized radioactivity represented <50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5-methyl[³H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37°C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[³H]THF was translocated to the cytosol and, despite differences in membrane levels of FRα expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FRα or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FRα activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5-methylTHF in these tumor cells depends not only on the level of overexpressed FRα, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake. J. Cell. Biochem. 65:479–491. © 1997 Wiley-Liss Inc.     

Chronic ethanol administration impairs the binding and endocytosis of asialo-orosomucoid in isolated hepatocytes

We have examined the effect of ethanol administration on receptor-mediated endocytosis of asialo-orosomucoid by isolated hepatocytes. Significantly less ligand was bound, internalized, and degraded by hepatocytes isolated from rats fed an ethanol diet for 5-7 weeks than by cells isolated from chow-fed or pair-fed controls. Reduced binding was shown to be primarily due to a decreased number of cell surface receptors rather than to a lowered affinity of the receptor for its ligand. This reduction in cell surface receptors resulted in a marked inhibition of internalization and degradation of ligand by hepatocytes from the ethanol-fed rats. In addition, a defect in the initial stages of receptor-ligand internalization was also indicated, since less surface-bound ligand was internalized and subsequently degraded in cells from the ethanol-treated animals as compared to controls. Rates of internalization and degradation of internalized ligand were, however, similar for all three groups, suggesting that neither degradation per se nor rate of delivery of internalized ligand to the lysosomes was affected by ethanol feeding. Receptor recycling was impaired in ethanol-fed rats, as indicated by a decrease in the binding site number after stimulation of endocytosis for 120 min when compared to initial binding capacity. Receptor recycling was not impaired in hepatocytes from control animals. These results indicate that chronic ethanol feeding impairs the process of receptor-mediated endocytosis by the liver; the major cause of this impairment appears to be due to a decreased number of cell surface asialoglycoprotein receptors in the ethanol-fed animals, along with a decreased ability of these cells to internalize all of the surface-bound ligand.     

Evaluation of the negative cooperativity model for fat cell beta-adrenergic receptors

Cooperative site-to-site interactions among beta-adrenergic receptors of fat cell membranes are probed with the potent beta-adrenergic antagonist (?)-[³H]-dihydroalprenolol according to the kinetic method of De Meyts et al. (De Meyts, P., Roth, J., Neville, Jr., D.M., Gavin, III, J.R. and Lesniak, M.A. (1973) Biochem. Biophys. Res. Commun. 55, 154–161). Dissociation of specific (?)-[³H]dihydroalprenolol binding from fat cell membranes following a 100-fold dilution was rapid at 37°C; only 40% of the initial equilibrium binding remained 30 s after dilution. Dissociation of (?)-[³H]dihydroalprenolol bound under conditions yielding approximately 20% initial occupancy was performed in the absence and in the presence of a large molar excess of beta-adrenergic agonist ((?)-isoproterenol) or beta-adrenergic antagonist ((?)-alprenolol or(?)-propanalol). Neither agonists nor antagonists influenced the rate of (?)-[³H]dihydroalprenolol dissociation from fat cell membranes performed at 4, 22 or 37°C. Although analysis of the steady-state binding of (?)-[³H]-dihydroalprenolol to fat cell membranes yields Hill coefficients, n_H, less than 1.0, the present study indicates that these fat cell beta-adrenergic receptors display no cooperative site-to-site interactions.     

Asialoglycoprotein receptor phosphorylation and receptor-mediated endocytosis in hepatoma cells. Effect of phorbol esters

The asialoglycoprotein (ASGP) receptor on Hep G2 cells undergoes constitutive recycling and ligand endocytosis in the presence of phorbol dibutyrate, at a 50% reduced rate relative to control cells (Fallon, R. J., and Schwartz, A. L. (1986) J. Biol. Chem. 261, 15081-15089). The relevance of receptor phosphorylation to these events was investigated by selective immunoprecipitation of surface receptors with polyclonal anti-human ASGP antiserum and pulse-chase labeling with [32P]orthophosphate to identify subcellular locations of initial receptor phosphorylation events as well as the eventual fate of phosphorylated receptor during recycling. The surface immunoprecipitation method recovers greater than 95% of surface ASGP receptors and only 5% or less of intracellular (brief[35S]methionine pulse-labeled) receptors. With this assay we detected low levels of ASGP receptor phosphorylation at the cell surface in control cells (0.1 mol of P/mol of R) which were rapidly (less than 1 min) stimulated 20-fold by 400 nM phorbol dibutyrate addition (1.7 mol of P/mol of R). Staurosporine, a protein kinase C inhibitor, blocks this stimulation by phorbol. Receptor phosphorylation at early time points in the presence of phorbol esters was restricted to the plasma membrane. Subsequent chase in the presence of excess unlabeled phosphate and phorbol esters lowered [32P] ATPi specific activity by 68% at 1 h. Surface immunoprecipitation during this chase period showed the phosphorylated ASGP receptors were rapidly lost from the cell surface (t1/2 = 20 min). In contrast, examination of intracellular receptor during the pulse-chase experiment in phorbol dibutyrate-treated cells showed the presence of phosphorylated pool(s) of ASGP receptors which were detectable for 6 h of chase. Since no labeled receptor can be detected at the cell surface at this time, the described intracellular phosphorylated receptors are in a non-recycling pool.     

Benzodiazepine receptors: temperature dependence of [3H]flunitrazepam binding.

The characteristics of [³H]flunitrazepam binding to brain specific benzodiazepine receptors were determined at varying temperatures. The rates at which [³H]flunitrazepam associated with and dissociated from benzodiazepine receptors increased with increasing temperatures. The dissociation constant (K_D) also increased with increases in temperature. The (K_D) determined by Scatchard analyses of saturation isotherms showed a similar change with changes in temperature. The maximal binding capacity (Bmax) did not change with changes in temperature. The inhibitory constants of several benzodiazepines to inhibit [³H]flunitrazepam binding to brain were also higher at 37°C than at 0°C, suggesting that the binding affinity of all benzodiazepines to brain benzodiazepine receptors is lower at 37°C than at 0°C. Van't Hoff analysis of [³H]flunitrazepam binding to brain at different temperatures reveals two linear components to this relationship.     

Rat hepatocytes bind to synthetic galactoside surfaces via a patch of asialoglycoprotein receptors

The binding of rat hepatocytes to flat polyacrylamide surfaces containing galactose is sugar-specific, requires Ca+2, and occurs only above a critical concentration of sugar in the substratum [Weigel et al., 1979, J. Biol. Chem., 254, 10,830). Binding is completely inhibited by asialo-orosomucoid but not by orosomucoid or asialo- agalacto-orosomucoid, suggesting that cell binding is mediated by asialoglycoprotein receptors. Asialo-orosomucoid was labeled with fluorescein isothiocyanate and used as a direct fluorescent probe to monitor the distribution of cell surface asialoglycoprotein receptors before and after hepatocyte binding to galactoside or control substrata. Cells bound at 37 degrees C were de-adhered at 4 degrees C using the Ca+2 chelator EGTA. The released cells were then stained with fluorescein-asialo-orosomucoid, fixed, washed, and examined by fluorescence microscopy. On freshly isolated cells before binding, the distribution of asialoglycoprotein receptors appears diffuse and nonclustered. However, more than half of the cells released intact from a galactoside surface had a single large (4 micrometer2) fluorescent patch. The receptor patch cannot be detected on cells while they are bound to a galactoside surface but rather only on released cells, indicating that the cell-substratum junction is the site of the receptor patch. No asialoglycoprotein receptor patches (less than or equal to 1%) were observed on cells that were incubated on, but did not bind to, an underivatized polyacrylamide surface or to a surface with a galactose concentration below the critical concentration for binding. Furthermore, no receptor patches were present on cells that had bound to and were subsequently released from substrata that did not contain galactose, including glass, tissue culture plastic, nontissue culture plastic, and collagen. The distribution of asialoglycoprotein receptors is preserved at 4 degrees C because at 37 degrees C the patches disappear with a half-life of approximately 2.6 min. The results directly demonstrate that a large cluster of asialoglycoprotein receptors mediates the binding of rat hepatocytes to a galactoside surface.     

Evidence for reutilization of surface receptors for alpha-macroglobulin.protease complexes in rabbit alveolar macrophages

Rabbit alveolar macrophages internalize α-macroglobulin ¹²⁵I-trypsin complexes subsequent to binding of complexes to high affinity surface receptors. Cells were capable of accumulating a 5–10 fold greater amount of αM · ¹²⁵I-T at 37°C than at 0°C. At 0°C cell-bound αM · ¹²⁵I-T was bound solely to surface receptors, whereas at 37°C the majority (85%) of cell-bound radioactivity was intracellular. The temperature-dependent accumulation of αM · ¹²⁵I-T did not reflect a change in surface receptor number or ligand-receptor affinity. Rather, the greater rate of uptake reflected continued internalization of αM · ¹²⁵I-T complexes. At 37°C cells took up 5–9 fmole αMT per μg cell protein per hr, whereas binding to surface receptors accounted for 0.5–0.7 fmole per μg cell protein. Once bound to surface receptors internalized αM · ¹²⁵I-T was localized in lysosomes, where it was degraded at a rate of 35–45% per hr. Following binding of αM · T to receptors at 37°C, but not at 0°C, unoccupied receptors could be found on the cell surface. Using cycloheximide to probe receptor turnover, I calculated that receptors were replenished at a rate of 15% per hr. Cells incubated in the presence of cycloheximide exhibited unaltered ligand uptake and catabolism for hours. Thus the reappearance of receptor activity during ligand uptake was not primarily due to de novo receptor synthesis. The rate of ligand uptake was a function of the number of surface receptors. Measurement of αM¹²⁵I-T binding to subcellular fractions did not reveal the presence of any intracellular reservoir of receptors. These observations are consistent with the hypothesis that continued ligand uptake reflects receptor reutilization.     

Differences in time course of internalization of receptors of insulin and insulin-like growth factor (IGF-1) in isolated rat hepatocytes

Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is a most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones at isolated rat hepatocytes, the internalization time course of ¹²⁵I-insulin and ¹²⁵I-IGF-I are traced at 37 and 12°C. There are established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37°C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. But essential differences in the internalization course of these two related peptides were obvious at the temperature of 12°C. The internalization level of insulin receptors at 12°C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocyte plasma membrane. At 12°C a slight decrease of the proportion of intracellular ¹²⁵I-IGF-I correlated with a decrease in the ¹²⁵I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12°C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar “inhibition mechanism” of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to action of cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates.     

The surface content of asialoglycoprotein receptors on isolated hepatocytes is reversibly modulated by changes in temperature

We have investigated the effect of temperature on the content of surface asialoglycoprotein receptors on isolated rat hepatocytes. Receptor was determined by measuring the specific binding of 125I- or [3H] asialo-orosomucoid at 0 degrees C. As reported previously, the receptor number/cell increases 2-3-fold within 30-60 min when freshly isolated cells are warmed from 0-37 degrees C (Weigel, P. H. (1980) J. Biol. Chem. 255, 6111-6120). This increase in receptor number is not inhibited by cycloheximide and also occurs on cells which have first been treated with EDTA to expose a population of cryptic receptors on the cell surface. The rate and extent of the receptor number increase on the cell surface are proportional to the temperature above about 17 degrees C. If cells are first equilibrated at 37 degrees C and then transferred to a lower temperature, the surface receptor number decreases at a rate and to an extent dependent on the temperature. The surface receptor number can be modulated up and down by successive temperature change cycles between 25 and 37 degrees C. In this temperature range, the number of surface receptors/cell is dependent on the final temperature but independent of the pathway to that temperature and is, therefore, a function of state with respect to temperature. The results demonstrate that temperature changes reversibly modulate the number of receptors on the hepatocyte surface. We conclude that, in the absence of ligand, surface receptors can either recycle or can be reversibly internalized or sequestered to prevent access to ligand. The results may also explain why different laboratories have reported a wide range of values for the number of receptors per hepatocyte.     

Internalization of insulin and its receptor in the isolated rat adipose cell: Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37°C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [¹²⁵I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [¹²⁵I]insulin to these same membrane fractions. At 37°C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20–30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16°C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.     

Thyroliberin is rapidly transferred to the nucleus of GH3 pituitary cells at both 4°C and 37°C

The time-course of thyroliberin transfer to the nucleus of GH3/B6 rat pituitary prolactin cells was studied by both autoradiography and cell fractionation of intact cells exposed to [³H]thyroliberin at 4°C or 37°C. It was previously shown that thyroliberin is not degraded in these conditions. It is found by autoradiography that [³H]-thyroliberin is transferred to the nucleus of GH3/B6 cells within 5 min at least at both 37° C and 4°C. Consistent results are obtained by fractionation of cells exposed to [³H]thyroliberin at 37°C. However after binding at 4°C 50% of the cell radioactivity is extractible by glutaraldehyde and after fractionation the isolated nuclei retain only 1–1.5% of the cell radioactivity. This suggests the existence of both tightly bound and loosely bound internalized thyroliberin molecules.     

Modulation by zinc chloride of concanavalin A binding to rat thymocytes and early inhibition of lectin-induced blastogenesis.

Zinc chloride was shown to modulate the number and mobility of concanavalin A receptors of rat thymocytes at 4 °C. Zinc was able to induce the exposition at the cell surface of the same number of receptors that can be exposed by high doses of concanavalin A. It only weakly restricted their mobility. Moreover, zinc chloride was shown to inhibit rat thymocyte stimulation by succinyl-concanavalin A. This inhibition occurred most effectively if zinc was present during the first few hours after mitogen addition to cell cultures, as assessed by [³H]uridine incorporation in RNA and [³H]thymidine incorporation in DNA. The time at which DNA synthesis took place was not modified by zinc. Blast transformation in the presence of zinc was characterized by fewer blast cells which otherwise appeared identical to blast cells in control cultures. The possible action on membrane-cytoskeleton interactions is discussed.     

The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions (Simons, T. J. B. (1979) Nature 281, 337-338). Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C (Weigel, P. H., and Oka, J. A. (1983) J. Biol. Chem. 258, 5089-5094); vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.